Bone morphogenetic protein (BMP)--6

ABSTRACT

Purified bone morphogenetic protein (BMP)-6 proteins and processes for producing them are disclosed. The proteins may be used in the treatment of bone and/or cartilage defects and in wound healing and related tissue repair.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/251,069 now U.S.Pat. No. 5,459,047 filed May 27, 1994, which is a divisional of U.S.Ser. No. 07/926,081 filed Aug. 5, 1992, now abandoned, which is adivisional of U.S. Ser. No. 07/490,033 now U.S. Pat. No. 5,187,076 filedMar. 7, 1990, which is a continuation-in-part of U.S. Ser. No.07/370,544 filed Jun. 23, 1989, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/347,559 filed May 4, 1989 (nowabandoned), which is a continuation-in-part of U.S. Ser. Nos. 07/179,100now U.S. Pat. No. 5,013,649; 07/179,101 (now abandoned); and 07/179,197filed Apr. 8, 1988, now abandoned, which are continuations-in-part ofU.S. Ser. Nos. 07/028,285 filed Mar. 20, 1987 (now abandoned); and07/031,346 now U.S. Pat. No. 4,877,864 filed Mar. 26, 1987, now U.S.Pat. No. 4,877,864, which are continuations-in-part of U.S. Ser. Nos.06/943,332 filed Dec. 17, 1986 (now abandoned;) and 06/880,776 filedJul. 1, 1986 (now abandoned).

The present invention relates to a family of purified proteins, termedBMP-6 proteins (wherein BMP is bone morphogenic protein), which exhibitthe ability to induce cartilage and/or bone formation and processes forobtaining them. These proteins may be used to induce bone and/orcartilage formation and in wound healing and tissue repair.

The invention provides purified human BMP-6 proteins, substantially freefrom other proteins with which they are co-produced. The BMP-6 proteinsof the invention are characterized by an amino acid sequence comprisingacid #412 to amino acid #513 set forth in FIG. 3. The amino acidsequence from amino acid #412 to #513 is encoded by the DNA sequence ofFIG. 3 from nucleotide #1393 to nucleotide #1698. These proteins may befurther characterized by an apparent molecular weight of 28,000-30,000daltons as determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Under reducing conditions in SDS-PAGE theprotein electrophoreses with a molecular weight of approximately14,000-20,000 daltons. It is contemplated that these proteins arecapable of stimulating promoting, or otherwise inducing cartilage and/orbone formation.

The invention, further provides bovine BMP-6 proteins characterized bythe amino acid sequence comprising amino acid #121 to amino acid #222set forth in FIG. 2. The amino acid sequence from #121 to #222 isencoded by the DNA sequence of FIG. 2 from nucleotide #361 to #666 ofFIG. 2. These proteins may be further characterized by an apparentmolecular weight of 28,000-30,000 daltons as determined by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Underreducing conditions in SDS-PAGE the protein electrophoreses with amolecular weight of approximately 14,000-20,000 daltons. It iscontemplated that these proteins are capable of inducing cartilageand/or bone formation.

Human BMP-6 proteins of the invention are produced by culturing a celltransformed with a DNA sequence comprising nucleotide #1393 tonucleotide #1698 as shown in FIG. 3 or a substantially similar sequence,recovering and purifying from the culture medium a protein comprisingamino acid #412 to amino acid #513 or a substantially similar sequence.

Bovine proteins of the invention may be produced by culturing a celltransformed with a DNA comprising nucleotide #361 through nucleotide#666 as set forth in FIG. 2 or a substantially similar sequence andrecovering and purifying from the culture medium a protein comprisingamino acid #121 to amino acid #222 as set forth in FIG. 2.

The invention further provides a method wherein the proteins describedabove are utilized for obtaining related human protein/s or othermammalian cartilage and/or bone formation protein/s. Such methods areknown to those skilled in the art of genetic engineering. One method forobtaining such proteins involves utilizing the human BMP-6 codingsequence or a portion thereof from nucleotide #160-#1698 to designprobes for screening human genomic and/or cDNA libraries to isolatehuman genomic and/or cDNA sequences. Additional methods within the artmay employ the bovine and human BMP-6 proteins of the invention toobtain other mammalian BMP-6 cartilage and/or bone formation proteins.Having identified the nucleotide sequences, the proteins are produced byculturing a cell transformed with the nucleotide sequence. This sequenceor portions thereof hybridizes under stringent conditions to thenucleotide sequence substantially as shown in Table II comprisingnucleotide #1 to nucleotide #666 or the nucleotide sequence or portionsthereof substantially as shown in FIG. 3 comprising nucleotide #160 to#1698 and encodes a protein exhibiting cartilage and/or bone formationactivity. The expressed protein is recovered and purified from theculture medium. The purified BMP-6 proteins of the invention aresubstantially free from other proteinaceous materials with which theyare coproduced, as well as from other contaminants.

The BMP-6 proteins of the invention are further characterized by theability to promote, stimulate or otherwise induce the formation ofcartilage and/or bone. It is further contemplated that the ability ofthese proteins to induce the formation of cartilage and/or bone isexhibited by the ability to demonstrate cartilage and/or bone formationactivity in the rat bone formation assay described below. It is furthercontemplated that the proteins of then demonstrate activity in this ratbone assay at a concentration of 10 μg-500 μg/gram of bone formed. Moreparticularly, it is contemplated these proteins may be characterized bythe ability of 1 μg of the protein to score at least +2 in the rat boneformation assay described below using either the original or modifiedscoring method.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a protein of theinvention in a pharmaceutically acceptable vehicle or carrier. Thesecompositions of the invention may be used to induce bone and/orcartilage formation. These compositions may also be used for woundhealing and tissue repair. Further compositions of the invention mayinclude, in addition to a BMP-6 protein, at least one othertherapeutically useful agent such as the proteins designated BMP-1,BMP-2A and -2B, BMP-3, BMP-5, and BMP-7 disclosed respectively inco-owned U.S. Pat. No. 5,013,649, Ser. No. 179,101, abandoned, and Ser.No. 179,197, abandoned U.S. Pat. No. 5,141,905, Ser. No. 438,919, nowU.S. Pat. No. 5,141,905. Other therapeutically useful agents includegrowth factors such as epidermal growth factor (EGF), fibroblast growthfactor (FGF), transforming growth factors (TGF-α and TGF-β) and plateletderived growth factor (PDGF). The compositions of the invention may alsoinclude an appropriate matrix, for instance, for delivery and support ofthe composition and/or providing a surface for bone and/or cartilagegrowth.

The compositions may be employed in methods for treating a number ofbone and/or cartilage defects, and periodontal disease. They may also beemployed in methods for treating various types of wounds and in tissuerepair. These methods, according to the invention, entail administeringto a patient needing such bone and/or cartilage formation, wound healingor tissue repair, a therapeutically effective amount of a BMP-6 proteinof the invention in a pharmaceutically acceptable vehicle or carrierincluding a martrix. These methods may also entail the administration ofa BMP-6 protein in conjunction with at least one of the "BMP" proteinsdisclosed in the co-owned applications described above. In addition,these methods may also include the administration of a protein of theinvention with other growth factors including EGF, FGF, TGF-α, TGF-β,and PDFG.

Still a further aspect of the invention are DNA sequences coding forexpression of a protein of the invention. Such sequences include thesequence of nucleotides in a 5' to 3' direction illustrated in FIG. 2 orFIG. 3 or DNA sequences which hybridize under stringent conditions withthe DNA sequence of FIG. 2 or FIG. III and encode a proteindemonstrating ability to induce cartilage and/or bone formation. Suchability to induce cartilage and/or bone formation may be demonstrated inthe rat bone formation assay described below. It is contemplated thatthese proteins demonstrate activity in this assay at a concentration of10 μg-500 μg/gram of bone formed. More particularly, it is contemplatedthat these proteins demonstrate the ability of 1 μg of the protein toscore at least +2 in the rat bone formation assay using either theoriginal or modified scoring method. Allelic or variations as describedherein below of the sequences of FIG. 2 and 3, whether such nucleotidechanges result in changes in the peptide sequence or not, are alsoincluded in the present invention.

A further aspect of the invention provides vectors containing a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing a protein of the invention in which a cell linetransformed with a DNA sequence directing expression of a protein of theinvention in operative association with an expression control sequencetherefor, is cultured in a suitable culture medium and a protein of theinvention is isolated and purified therefrom. This claimed process mayemploy a number of known cells, both prokaryotic and eukaryotic, as hostcells for expression of the polypeptide.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description and preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 comprises DNA sequence and derived amino acid sequence of bovineBMP-5.

FIG. 2 comprises DNA sequence and derived amino acid sequence of bovineBMP-6.

FIG. 3 comprises DNA sequence and derived amino acid sequence of humanBMP-6 from BMP6C35 ATCC #68245.

DETAILED DESCRIPTION OF THE INVENTION

The purified human BMP-6 proteins of the invention are characterized byan amino acid sequence comprising amino acid #412 to #513 as set forthin FIG. 3. In one embodiment a BMP-6 protein of the invention comprisesamino acid #388 to #513 of FIG. 3. In a further embodiment the BMP-6protein comprises amino acid #382 to #513 of FIG. 3.

The purified BMP-6 human cartilage/bone proteins of the presentinvention may be produced by culturing a host cell transformed with aDNA sequence comprising nucleotide #1393 to nucleotide #1698 as setforth in FIG. 3 or substantially homologous sequences operatively linkedto a heterologous regulatory control sequence and recovering, isolatingand purifying from the culture medium a protein comprising amino acid#412 to amino acid #513 as set forth in FIG. 3 or a substantiallyhomologous sequence.

In another embodiment, purified human BMP-6 proteins may be produced byculturing a host cell transformed with a DNA sequence comprisingnucleotide #1321 to #1698 as set forth in FIG. 3 or substantiallyhomologous sequences operatively linked to a heterologous regulatorycontrol sequence and recovering and purifying from the culture medium aprotein comprising amino acid #388 to #513 asset forth in FIG. 3 or asubstantially homologous sequence.

In another embodiment, purified human BMP-6 proteins may be produced byculturing a host cell transformed with a DNA sequence comprisingnucleotide #1303 to #1698 as set forth in FIG. 3 or substantiallyhomologous sequences operatively linked to a heterologous regulatorycontrol sequence and recovering and purifying from the culture medium aprotein comprising amino acid #382 to #513 as set forth in FIG. 3 or asubstantially homologous sequence.

The purified human BMP-6 proteins are substantially free from otherproteinaceous materials with which they are co-produced, as well as fromother contaminants.

Purified BMP-6 bovine cartilage/bone protein of the present inventionare produced by culturing a host cell transformed with a DNA sequencecomprising nucleotide #361 to nucleotide #666 as set forth in FIG. 2 orsubstantially homologous sequences and recovering from the culturemedium a protein comprising amino acid #121 to amino acid #222 as setforth in FIG. 2 or a substantially homologous sequence. In anotherembodiment the bovine protein is produced by culturing a host celltransformed with a sequence comprising nucleotide #289 to #666 of FIG. 2and recovering and purifying a protein comprising amino acid #97 toamino acid #222. The purified BMP-6 bovine proteins are substantiallyfree from other proteinaceous materials with which they are co-produced,as well as from other contaminants.

These proteins of the invention may be further characterized by theability to demonstrate cartilage and/or bone formation activity. Thisactivity may be demonstrated, for example, in the rat bone formationassay as described in Example III. It is further contemplated that theseproteins demonstrate activity in the assay at a concentration of 10μg-500 μg/gram of bone formed. The proteins may be further characterizedby the ability of 1 μg to score at least +2 in this assay.

BMP-6 proteins may be further characterized by an apparent molecularweight of 28,000-30,000 daltons as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditionsin SDS-PAGE, the protein electrophoreses with a molecular weight ofapproximately 14,000-20,000 daltons.

The proteins provided herein also include factors encoded by thesequences similar to those of FIG. 2 and FIG. 3 but into whichmodifications are naturally provided (e.g. allelic variations in thenucleotide sequence which may result in amino acid changes in thepolypeptide) or deliberately engineered. Similarly, syntheticpolypeptides which wholly or partially duplicate continuous sequences ofthe amino acid residues of FIG. 2 or FIG. 3 are encompassed by theinvention. These sequences, by virtue of sharing primary, secondary, ortertiary structural and conformational characteristics with othercartilage/bone proteins of the invention may possess bone and/orcartilage growth factor biological properties in common therewith. Thus,they may be employed as biologically active substitutes fornaturally-occurring proteins in therapeutic processes.

Other specific mutations of the sequences of the proteins of theinvention described herein may involve modifications of a glycosylationsite. These modifications may involve O-linked or N-linked glycosylationsites. For instance, the absence of glycosylation or only partialglycosylation at the asparagine-linked glycosylation sites results fromamino acid substitution or deletion at the asparagine-linkedglycosylation recognition sites present in the sequences of the proteinsof the invention, for example, as shown in FIG. 2 or FIG. 3. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are eitherasparagine-X-threonine or asparagine-X-serine, where X is usually anyamino acid. A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.Expression of such altered nucleotide sequences produces variants whichare not glycosylated at that site.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding on expression for the proteins of the invention. These DNAsequences include those depicted in FIGS. 2 and 3 in a 5' to 3'direction or portions thereof. Further included are those sequenceswhich hybridize under stringent hybridization conditions see, T.Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold SpringHarbor Laboratory (1982), pages 387 to 389! to the DNA sequence of FIG.2 or FIG. 3 and demonstrate cartilage and/or bone formation activity.Such cartilage and/or bone formation activity may be in the rat boneformation assay. An example of one such stringent hybridizationcondition is hybridization at 4× SSC at 65° C., followed by a washing in0.1× SCC at 65° C. for an hour. Alternatively, an exemplary stringenthybridization condition is in 50% formamide, 4× SCC at 42° C.

Similarly, DNA sequences which encode proteins similar to the proteinencoded by the sequence of FIG. 2 or FIG. 3, but which differ in codonsequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode theproteins of the invention described herein. Variations in the DNAsequences of FIG. 2 and FIG. 3 which are caused by point mutations or byinduced modifications (including insertion, deletion, and substitution)to enhance the activity, half-life or production of the polypeptidesencoded thereby are also encompassed in the invention.

In a further aspect, the invention provides a method for obtainingrelated human proteins or other mammalian BMP-6 proteins. One method forobtaining such proteins entails, for instance, utilizing the human BMP-6coding sequence disclosed herein to probe a human genomic library usingstandard techniques for the human gene or fragments thereof. Sequencesthus identified may also be used as probes to identify a human cell lineor tissue which synthesizes the analogous cartilage/bone protein. A cDNAlibrary is synthesized and screened with probes derived from the humanor bovine coding sequences. The human sequence thus identified istransformed into a host cell, the host cell is cultured and the proteinrecovered, isolated and purified from the culture medium. The purifiedprotein is predicted to exhibit cartilage and/or bone formationactivity. This activity may be demonstrated in the rat bone formationassay of Example III.

Another aspect of the present invention provides a novel method forproducing the proteins of the invention. This method involves culturinga suitable cell line, which has been transformed with a DNA sequencecoding for expression of a protein of the invention, under the controlof known regulatory sequences. Regulatory sequences include promoterfragments, terminator fragments and other suitable sequences whichdirect the expression of the protein in an appropriate host cell.Methods for culturing suitable cell lines are within the skill of theart. The transformed cells are cultured and the BMP-6 proteins expressedthereby are recovered and purified from the culture medium usingpurification techniques known to those skilled in the art. The purifiedBMP-6 proteins are substantially free from other proteinaceous materialswith which they are co-produced, as well as other contaminants. PurifiedBMP-6 proteins of the invention are substantially free from materialswith which the proteins of the invention exist in nature.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screeningand product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al,U.S. Pat. No. 4,419,446. Other suitable mammalian cell lines are themonkey COS-1 cell line and the CV-1 cell line.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of the proteins of the invention. Preferably thevectors contain the full novel DNA sequences described above which codefor the novel BMP-6 proteins of the invention. Additionally the vectorsalso contain appropriate expression control sequences permittingexpression of the protein sequences. Alternatively, vectorsincorporating modified sequences as described above are also embodimentsof the present invention and useful in the production of the proteins ofthe invention. The vectors may be employed in the method of transformingcell lines and contain selected regulatory sequences in operativeassociation with the DNA coding sequences of the invention which arecapable of directing the replication and expression thereof in selectedhost cells. Useful regulatory sequences for such vectors are known tothose skilled in the art and may be selected depending upon the selectedhost cells. Such selection is routine and does not form part of thepresent invention. Host cells transformed with such vectors and progenythereof for use in producing BMP-6 proteins are also provided by theinvention.

A protein of the present invention, which induces cartilage and/or boneformation in circumstances where bone and/or cartilage is not normallyformed, has application in the healing of bone fractures and cartilagedefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. A protein of the invention may be used in the treatmentof periodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A variety of osteogenic, cartilage-inducing and boneinducing factors have been described. See, e.g. European patents 148,155and 169,016 for discussions thereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair).

A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related to boneand/or cartilage defects or periodontal diseases. In addition, theinvention comprises therapeutic methods and compositions for woundhealing and tissue repair. Such compositions comprise a therapeuticallyeffective amount of at least one of the BMP-6 proteins of the inventionin admixture with a pharmaceutically acceptable vehicle, carrier ormatrix.

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with one another or with other relatedproteins and growth factors. Therapeutic methods and compositions of theinvention therefore comprise one or more of the proteins of the presentinvention. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one protein of theinvention with a therapeutic amount of at least one of the other "BMP"proteins, BMP-1, BMP-2 (BMP-2A, BMP-2 Class I), BMP-3, BMP-4 (BMP-2B,BMP-2 Class II), BMP-5 and BMP-7, disclosed in co-owned and co-pendingU.S. applications described above. Such methods and compositions of theinvention may comprise proteins of the invention or portions thereof incombination with the above-mentioned "BMP" proteins or portions thereof.Such combination may comprise individual separate molecules from each ofthe proteins or heteromolecules such as heterodimers formed by portionsof the respective proteins. For example, a method and composition of theinvention may comprise a BMP-6 protein of the invention or a portionthereof linked with a portion of a different "BMP" protein to form aheteromolecule.

Further therapeutic methods and compositions of the invention comprisethe proteins of the invention or portions thereof in combination withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), fibroblast growthfactor (FGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), k-fibroblast growth factor (kFGF),parathyroid hormone (PTH), leukemia inhibitory factor (LIF/HILDA/DIA),and insulin-like growth factors (IGF-I and IGF-II). Portions of theseagents may also be used in compositions of the invention.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the apparent lack of species specificity in cartilage and boneproteins. Domestic animals and thoroughbred horses in addition to humansare desired patients for such treatment with the proteins of the presentinvention.

The therapeutic method includes administering the composition topically,systematically, or locally as an implant or device. When administered,the therapeutic composition for use in this invention is, of course, ina pyrogen-free, physiologically acceptable form. Further, thecomposition may desirably be encapsulated or injected in a viscous formfor delivery to the site of cartilage and/or bone or tissue damage.Topical administration may be suitable for wound healing and tissuerepair. Preferably for bone and/or cartilage formation, the compositionincludes a matrix capable of delivering the cartilage/bone proteins ofthe invention to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and optimally capable ofbeing resorbed into the body. Matrices may provide slow release of thecartilage and/or bone inductive proteins proper presentation andappropriate enviroment for cellular infiltration. Matrices may be formedof materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositions ofthe invention will define the appropriate formulation. Potentialmatrices for the compositions are biodegradable and chemically definedcalcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acidand polyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the proteins ofthe invention. Factors which may modify the action of the proteins ofthe invention include the amount of bone weight desired to be formed,the site of bone damage, the condition of the damaged bone, the size ofa wound, type of damaged tissue, the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and the type or types of bone and/or cartilage proteinspresent in the composition. The addition of other known growth factors,such as EGF, PDGF, TGF-α, TGF-β, and IGF-I to the final composition, mayalso effect the dosage.

Progress can be monitored by periodic assessment of cartilage and/orbone growth and/or repair. The progress can be monitored, for example,using x-rays, histomorphometric determinations and tetracyclinelabeling.

The following examples illustrate practice of the present invention inrecovering and characterizing bovine cartilage and/or bone proteins ofthe invention and employing these proteins to recover the correspondinghuman protein or proteins and in expressing the proteins via recombinanttechniques.

EXAMPLE I Isolation of Bovine Cartilage/Bone Inductive Protein

Ground bovine bone powder (20-120 mesh, Helitrex) is prepared accordingto the procedures of M. R. Urist et al., Proc. Natl Acad. Sci USA,70:3511 (1973) with elimination of some extraction steps as identifiedbelow. Ten kgs of the ground powder is demineralized in successivechanges of 0.6N HCl at 41/2° C. over a 48 hour period with vigorousstirring. The resulting suspension is extracted for 16 hours at 41/2° C.with 50 liters of 2M CaCl₂ and 10 mM ethylenediamine-tetraacetic acidEDTA!, and followed by extraction for 4 hours in 50 liters of 0.5M EDTA.The residue is washed three times with distilled water before itsresuspension in 20 liters of 4M guanidine hydrochloride GuCl!, 20 mMTris (pH 7.4), 1 mM N-ethylmaleimide, 1 mM iodoacetamide, 1 mMphenylmethylsulfonyl fluorine as described in Clin. Orthop. Rel. Res.,171: 213 (1982). After 16 to 20 hours the supernatant is removed andreplaced with another 10 liters of GuCl buffer. The residue is extractedfor another 24 hours.

The crude GuCl extracts are combined, concentrated approximately 20times on a Pellicon apparatus with a 10,000 molecular weight cut-offmembrane, and then dialyzed in 50 mm Tris, 0.1M NaCl, 6M urea (pH7.2),the starting buffer for the first column. After extensive dialysis theprotein is loaded on a 4 liter DEAE cellulose column and the unboundfractions are collected.

The unbound fractions are concentrated and dialyzed against 50 mM NaAc,50 mM NaCl (pH 4.6) in 6M urea. The unbound fractions are applied to acarboxymethyl cellulose column. Protein not bound to the column isremoved by extensive washing with starting buffer, and the materialcontaining protein having bone and/or cartilage formation activity asmeasured by the Rosen-modified Sampath--Reddi assay (described inExample III below) desorbed from the column by 50 mM NaAc, 0.25 mM NaCl,6M urea (pH 4.6). The protein from this step elution is concentrated 20-to 40-fold, then diluted 5 times with 80 mM KPO₄, 6M urea (pH6.0). ThepH of the solution is adjusted to 6.0 with 500 mM K₂ HPO₄. The sample isapplied to an hydroxylapatite column (LKB) equilibrated in 80 mM KPO₄,6M urea (pH6.0) and all unbound protein is removed by washing the columnwith the same buffer. Protein having bone and/or cartilage formationactivity is eluted with 100 mM KPO₄ (pH7.4) and 6M urea.

The protein is concentrated approximately 10 times, and solid NaCl addedto a final concentration of 0.15M. This material is applied to aheparin--Sepharose column equilibrated in 50 mM KPO₄, 150 mM NaCl, 6Murea (pH7.4). After extensive washing of the column with startingbuffer, a protein with bone and/or cartilage inductive activity iseluted by 50 mM KPO₄, 700 mM NaCl, 6M urea (pH7.4). This fraction isconcentrated to a minimum volume, and 0.4 ml aliquots are applied toSuperose 6 and Superose 12 columns connected in series, equilibratedwith 4M GuCl, 20 mM Tris (pH7.2) and the columns developed at a flowrate of 0.25 ml/min. The protein demonstrating bone and/or cartilageinductive activity corresponds to an approximate 30,000 dalton protein.

The above fractions from the superose columns are pooled, dialyzedagainst 50 mM NaAc, 6M urea (pH4.6), and applied to a Pharmacia MonoS HRcolumn. The column is developed with a gradient to 1.0M NaCl, 50 mMNaAc, 6M urea (pH4.6). Active bone and/or cartilage formation fractionsare pooled. The material is applied to a 0.46×25 cm Vydac C4 column in0.1% TFA and the column developed with a gradient to 90% acetonitrile,0.1% TFA (31.5% acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFAin 60 minutes at 1 ml per minute). Active material is eluted atapproximately 40-44% acetonitrile. Fractions were assayed for cartilageand/or bone formation activity. The active material is furtherfractionated on a MonoQ column. The protein is dialyzed against 6M urea,25 mM diethanolamine, pH 8.6 and then applied to a 0.5 by 5 cm MonoQcolumn (Pharmacia) which is developed with a gradient of 6M urea, 25 mMdiethanolamine, pH 8.6 and 0.5M NaCl, 6M urea, 25 mM diethanolamine, pH8.6. Fractions are brought to pH3.0 with 10% trifluoroacetic acid (TFA).

Aliquots of the appropriate fractions are iodinated by one of thefollowing methods: P. J. McConahey et al, Int. Arch. Allergy, 29:185-189(1966); A. E. Bolton et al, Biochem J., 133:529 (1973); and D. F.Bowen-Pope, J. Biol. Chem., 237:5161 (1982). The iodinated proteinspresent in these fractions are analyzed by SDS gel electrophoresis.

EXAMPLE II Characterization of Bovine Cartilage/Bone Inductive Factor

A. Molecular Weight

Approximately 5 μg protein from Example I in 6M urea, 25 mMdiethanolamine, pH 8.6, approximately 0.3M NaCl is made 0.1% withrespect to SDS and dialyzed against 50 mM tris/HCl 0.1% SDS pH 7.5 for16 hrs. The dialyzed material is then electrophorectically concentratedagainst a dialysis membrane Hunkapillar et al Meth. Enzymol. 91: 227-236(1983)! with a small amount of I 125 labelled counterpart. This material(volume approximately 100 μl) is loaded onto a 12% polyacrylamide geland subjected to SDS-PAGE Laemmli, U.K. Nature, 227:680-685 (1970)!without reducing the sample with dithiothreitol. The molecular weight isdetermined relative to prestained molecular weight standards (BethesdaResearch Labs). Following autoradiography of the unfixed gel theapproximate 28,000-30,000 dalton band is excised and the proteinelectrophoretically eluted from the gel (Hunkapillar et al supra). Basedon similar purified bone fractions as described in the co-pending "BMP"applications described above wherein bone and/or cartilage activity isfound in the 28,000-30,000 region, it is inferred that this bandcomprises bone and/or cartilage inductive fractions.

B. Subunit Characterization

The subunit composition of the isolated bovine bone protein is alsodetermined. The eluted protein described above is fully reduced andalkylated in 2% SDS using iodoacetate and standard procedures andreconcentrated by electrophoretic packing. The fully reduced andalkylated sample is then further submitted to SDS-PAGE on a 12% gel andthe resulting approximate 14,000-20,000 dalton region having a doubletappearance located by autoradiography of the unfixed gel. A faint bandremains at the 28,000-30,000 region. Thus the 28,000-30,000 daltonprotein yields a broad region of 14,000-20,000 which may otherwise alsobe interpreted and described as comprising two broad bands ofapproximately 14,000-16,000 and 16,000-20,000 daltons.

EXAMPLE III Rosen Modified Sampath-Reddi Assay

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. U.S.A., 80:6591-6595 (1983) is used toevaluate bone and/or cartilage activity of the proteins of theinvention. This modified assay is herein called the Rosen-modifiedSampath-Reddi assay. The ethanol precipitation step of the Sampath-Reddiprocedure is replaced by dialyzing (if the composition is a solution) ordiafiltering (if the composition is a suspension) the fraction to beassayed against water. The solution or suspension is then redissolved in0.1% TFA, and the resulting solution added to 20 mg of rat matrix. Amock rat matrix sample not treated with the protein serves as a control.This material is frozen and lyophilized and the resulting powderenclosed in #5 gelatin capsules. The capsules are implantedsubcutaneously in the abdominal thoracic area of 21-49 day old male LongEvans rats. The implants are removed after 5-21 days. Half of eachimplant is used for alkaline phosphatase analysis See, A. H. Reddi etal., Proc. Natl Acad Sci., 69:1601 (1972)!.

The other half of each implant is fixed and processed for histologicalanalysis. Glycolmethacrylate sections (1 μm) are stained with Von Kossaand acid fuschin or toluidine blue to score the amount of induced boneand cartilage formation present in each implant. The terms +1 through +5represent the area of each histological section of an implant occupiedby new bone and/or cartilage cells and newly formed bone and matrix. Twoscoring methods are herein described. The first describes the originalscoring method while the second describes the later adopted scoringmethod. A score of +5 indicates that greater than 50% of the implant isnew bone and/or cartilage produced as a direct result of protein in theimplant. A score of +4, +3, +2 and +1 would indicate that greater than40%, 30%, 20% and 10% respectively of the implant contains new cartilageand/or bone. The scoring method later adopted (which herein after maybereferred to as the "modified" scoring method) is as follows: Threenon-adjacent sections are evaluated from each implant and averaged."+/-" indicates tentative identification of cartilage or bone;"+2", >25%; "+3+", >50%; "+4", >75%; "+5", >80%. The scores of theindividual implants are tabulated to indicate assay variability.

It is contemplated that the dose response nature of the cartilage and/orbone inductive protein containing samples of the matrix samples willdemonstrate that the amount of bone and/or cartilage formed increaseswith the amount of cartilage/bone inductive protein in the sample. It iscontemplated that the control samples will not result in any bone and/orcartilage formation.

As with other cartilage and/or bone inductive proteins such as theabove-mentioned "BMP" proteins, the bone and/or cartilage formed isexpected to be physically confined to the space occupied by the matrix.Samples are also analyzed by SDS gel electrophoresis and isoelectricfocusing followed by autoradiography. The activity is correlated withthe protein bands and pI. To estimate the purity of the protein in aparticular fraction an extinction coefficient of 1 OD/mg-cm is used asan estimate for protein and the protein is run on SDS PAGE followed bysilver staining or radioiodination and autoradiography.

EXAMPLE IV Bovine BMP-6 Protein Composition

The gel slice of the approximate 14,000-20,000 dalton region describedin Example IIB is fixed with methanol-acetic acid-water using standardprocedures, briefly rinsed with water, then neutralized with 0.1Mammonium bicarbonate. Following dicing the gel slice with a razor blade,the protein is digested from the gel matrix by adding 0.2 μg ofTPCK-treated trypsin (Worthington) and incubating the gel for 16 hr. at37 degrees centigrade. The resultant digest is then subjected to RPHPLCusing a C4 Vydac RPHPLC column and 0.1% TFA-water 0.1% TFAwater-acetonitrile gradient. The resultant peptide peaks were monitoredby UV absorbance at 214 and 280 nm and subjected to direct aminoterminal amino acid sequence analysis using an Applied Biosystems gasphase sequenator (Model 470A). one tryptic fragment is isolated bystandard procedures having the following amino acid sequence asrepresented by the amino acid standard three-letter symbols and where"Xaa" indicates an unknown amino acid the amino acid in parenthesesindicates uncertainty in the sequence:

Xaa-His-Glu-Leu-Tyr-Val-Ser-Phe-(Ser)

The following four oligonucleotide probes are designed on the basis ofthe amino acid sequence of the above-identified tryptic fragment andsynthesized on an automated DNA synthesizer.

PROBE #1: GTRCTYGANATRCANTC

PROBE #2: GTRCTYGANATRCANAG

PROBE #3: GTRCTYAAYATRCANTC

PROBE #4: GTRCTYAAYATRCANAG

The standard nucleotide symbols in the above identified probes are asfollows: A,adenine; C,cytosine; G,guanine; T,thymine; N, adenosine orcytosine or guanine or thymine, R,adenosine or guanine; and Y,cytosineor thymine.

Each of the probes consists of pools of oligonucleotides. Because thegenetic code is degenerate (more than one codon can code for the sameamino acid), a mixture of oligonucleotides is synthesized that containsall possible nucleotide sequences encoding the amino acid sequence ofthe tryptic fragment. These probes are radioactively labeled andemployed to screen a bovine cDNA library as described below.

Poly(A) containing RNA is isolated by oligo(dT) cellulose chromatographyfrom total RNA isolated from fetal bovine bone cells by the method ofGehron-Robey et al in Current Advances in Skeletogenesis, ElsevierScience Publishers (1985). The total RNA was obtained from Dr. MarianYoung, National Institute of Dental Research, National Institutes ofHealth. A cDNA library is made in lambda gt10 (Toole et al supra) andplated on 50 plates at 8000 recombinants per plate. These recombinants(400,000) are screened on duplicate nitrocellulose filters with acombination of Probes 1, 2, 3, and 4 using the Tetramethylammoniumchloride (TMAC) hybridization procedure see Wozney et al Science, 242:1528-1534 (1988)!. Twenty-eight positives are obtained and are replatedfor secondaries. Duplicate nitrocellulose replicas again are made. Oneset of filters are screened with Probes 1 and 2; the other with Probes 3and 4. Six positives are obtained on the former, 21 positives with thelatter. One of the six, called HEL5, is plaque purified, a phage platestock made, and bacteriophage DNA isolated. This DNA is digested withEcoRI and subcloned into M13 and pSP65. The DNA sequence and derivedamino acid sequence of this fragment is shown in Table I.

DNA sequence analysis of this fragment in M13 indicates that it encodesthe desired tryptic peptide sequence set forth above, and this derivedamino acid sequence is preceded by a basic residue (Lys) as predicted bythe specificity of trypsin. The underlined portion of the sequence inFIG. 1 from amino acid #42 to #48 corresponds to the tryptic fragmentidentified above from which the oligonucleotide probes are designed. Thederived amino acid sequence Ser-Gly-Ser-His-Gln-Asp-Ser-Ser-Arg as setforth in FIG. 1 from amino acid #15 to #23 is noted to be similar to atryptic fragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found inthe 28,000-30,000 dalton purified bone preparation as described in the"BMP" co-pending applications mentioned above. This fragment set forthin FIG. 1 is a portion of the DNA sequence which encodes a bovine BMP-5protein. The DNA sequence indicates an open reading frame from the 5'end of the clone of 420 base pairs, encoding a partial peptide of 140amino acid residues (the first 7 nucleotides are of the adaptors used inthe cloning procedure). An in-frame stop codon (TAA) indicates that thisclone encodes the carboxy-terminal part of a bovine BMP-5 cartilage/boneprotein.

The remaining positive clones isolated with probes #1, #2, #3, and #4described above are screened with HEL5 and a further clone is identifiedunder reduced stringency conditions 5× SSC, 0.1% SDS, 5× Denhardt's, 100μg/ml salmon sperm DNA standard hybridization buffer (SHB) at 65° C.,wash in 2× SSC 0.1% SDS at 65° C.!. This clone is plaque purified, aphage plate stock made and bacteriophage DNA isolated. The DNA sequenceand derived amino acid sequence of a portion of this clone is shown inFIG. 2. This sequence represents the DNA sequence encoding a BMP-6cartilage/bone protein of the invention.

The first underlined portion of the sequence in FIG. 2 from amino acid#97-amino acid #105 Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg corresponds tothe tryptic fragment found in the 28,000-30,000 dalton purified bovinebone preparation (and its reduced form at approximately 18,000-20,000dalton reduced form) as described in the "BMP" co-pending applicationsmentioned above. The second underlined sequence in FIG. 2 from aminoacid #124-amino acid #130 corresponds to the tryptic fragment identifiedabove from which the oligonucleotide probes are designed.

The DNA sequence of FIG. 2 indicates an open reading frame of 666 basepairs starting from the 5' end of the sequence of FIG. 2, encoding apartial peptide of 222 amino acid residues. An in-frame stop codon (TGA)indicates that this clone encodes the carboxy-terminal part of a bovineBMP-6 protein of the invention. Based on knowledge of other BMP proteinsand other proteins in the TGF-β family, it is predicted that theprecursor polypeptide would be cleaved at the three basic residues(ArgArgArg) to yield a mature peptide beginning with residue 90 or 91 ofthe sequence of FIG. 2.

EXAMPLE V Human BMP-6 Proteins

Human cell lines which synthesize BMP-5 and/or BMP-6 mRNAs areidentified in the following manner. RNA is isolated from a variety ofhuman cell lines, selected for poly(A)-containing RNA by chromatographyon oligo(dT) cellulose, electrophoresed on a formaldehyde-agarose gel,and transferred to nitrocellulose. A nitrocellulose replica of the gelis hybridized to a single standed M13 ³² P-labeled probe correspondingto the above mentioned BMP-5 EcoRI-BglII fragment containing nucleotides1-465 of the sequence of Table I. A strongly hybridizing band isdetected in the lane corresponding to the human osteosarcoma cell lineU-20S RNA. Another nitrocellulose replica is hybridized to a singlestranded M13 ³² P-labeled probe containing the PstI-SmaI fragment ofbovine BMP-6 (corresponding to nucleotides 106-261 of FIG. 2. It isfound that several RNA species in the lane corresponding to U-20S RNAhybridize to this probe.

A cDNA Library is made in the vector lambda ZAP (Stratagene) from U-20Spoly(A)-containing RNA using established techniques (Toole et al.).750,000 recombinants of this library are plated and duplicatenitrocellulose replicas made. The SmaI fragment of bovine BMP-6corresponding to nucleotides 259-751 of FIG. 2 is labeled bynick-translation and hybridized to both sets of filters in SHB at 65°.One set of filters is washed under stringent conditions (0.2× SSC, 0.1%SDS at 65°), the other under reduced stringency conditions (1× SSC, 0.1%SDS at 65°). Many duplicate hybridizing recombinants (approximately 162)are noted. 24 are picked and replated for secondaries. Threenitrocellulose replicas are made of each plate. One is hybridized to theBMP-6 SmaI probe, one to a nick-translated BMP-6 PstI-SacI fragment(nucleotides 106-378 of FIG. 2), and the third to the nick-translatedBMP-5 XbaI fragment (nucleotides 1-76 of FIG. 1). Hybridization andwashes are carried out under stringent conditions.

Six clones which hybridize to the second probe more strongly than to thethird are picked and transformed into plasmids. Restriction mapping,Southern blot analysis, and DNA sequence analysis of these plasmidsindicate that there are two classes of clones. Clones U2-7 and U2-10contain human BMP-6 coding sequence based on their strongerhybridization to the second probe and closer DNA homology to the bovineBMP-6 sequence of FIG. 2 than the other 4 clones. DNA sequence dataderived from these clones indicates that they encode a partialpolypeptide of 132 amino acids comprising the carboxy-terminus of thehuman BMP-6 protein. U2-7 was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. on Jun. 23,1989 under accession number 68021.

A primer extended cDNA library is made from U-2 OS mRNA using theoligonucleotide GGAATCCAAGGCAGAATGTG, the sequence being based on the 3'untranslated sequence of the human BMP-6 derived from the clone U2-10.This library is screened with an oligonucleotide of the sequenceCAGAGTCGTAATCGC, derived from the BMP-6 coding sequence of U2-7 andU2-10. Hybridization is in standard hybridization buffer (SHB) at 42degrees centigrade, with wash conditions of 42 degrees centigrade, 5×SSC, 0.1% SDS. Positively hybridizing clones are isolated. The DNAinsert of one of these clones, PEH6-2 indicates that it extends furtherin a 5' direction than either U2-7 or U2-10. A primer extended cDNAlibrary constructed from U-20S mRNA as above is screened with anoligonucleotide of the sequence GCCTCTCCCCCTCCGACGCCCCGTCCTCGT, derivedfrom the sequence near the 5' end of PEH6-2. Hybridization is at 65degrees centigrade in SHB, with washing at 65 degrees centigrade in 2×SSC, 0.1% SDS. Positively hybridizing recombinants are isolated andanalyzed by restriction mapping and DNA sequence analysis.

The 5' sequence of the insert of one of the positively hybridizingrecombinants, PE5834#7, is used to design an oligonucleotide of thesequence CTGCTGCTCCTCCTGCTGCCGGAGCGC. A random primed cDNA librarysynthesized as for an oligo (dT) primed library except that (dN)₆ isused as the primer! is screened with this oligonucleotide byhybridization at 65 degrees centigrade in SHB with washing at 65 degreescentigrade in 1× SSC, 0.1% SDS. A positively hybridizing clone, RP10, isidentified, isolated, and the DNA sequence sequence from the 5' end ofits insert is determined. This sequence is used to design anoligonucletide of the sequenceTCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGGAGATGCA. A human placentacDNA library (Stratagene catalog #936203) is screened with thisoligonucleotide by hybridization in SHB at 65 degrees centigrade, andwashing at 65 degrees centigrade with 0.2× SSC, 0.1% SDS. A positivelyhybridizing recombinant designated BMP6C35 is isolated. DNA sequenceanalysis of the insert of this recombinant indicates that it encodes thecomplete human BMP-6 protein. BMP6C35 was deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va. USAon Mar. 1, 1990 under Accession Number 68245.

The DNA and derived amino acid sequence of the majority of the insert ofBMP6C35 is given in FIG. 3. This DNA sequence contains an open readingframe of 1539 base pairs which encodes the 513 amino acid human BMP-6protein precursor. The presumed initiator methionine codon is precededby a 5'untranslated sequence of 159 base pairs with stop codons in allthree reading frames. The stop codon at nucleotides 1699-1701 isfollowed by at least 1222 base pairs of 3'untranslated sequence. It isnoted that U2-7 has a C residue at the position corresponding to the Tresidue at position 1221 of BMP6C35; U2-7 also has a C residue at theposition corresponding to the G residue at position 1253 of BMP6C35.These do not cause amino acid differences in the encoded proteins, andpresumably represent allelic variations.

The oligonucleotide TCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGGAGATGCAis used to screen a human genomic library (Toole et al supra), byhybridizing nitrocellulose replicas of 1×10⁶ recombinants with theoligonucleotide in SHB at 65 degrees centigrade, and washing at 65degrees centigrade with 0.2× SSC, 0.1% SDS. Positively hybridizingclones are purified. The oligonucleotide hybridizing region is localizedto an approximately 1.5 kb Pst I fragment. DNA sequence analysis of thisfragment confirms the 5' sequence indicated in FIG. 3.

The DNA sequence of the human BMP-6 clone set forth in FIG. 3 reveals anopen reading frame of 1539 bp encoding a protein of 513 amino acids. Thefirst underlined portion of the sequence in FIG. 3 from amino acid #388to #396,Ser-Thr-Gln-Ser-Gln-Asp-Val-Ala-Arg, corresponds to the similarsequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg of the bovine sequencedescribed above and set forth in FIG. 2. The second underlined sequencein FIG. 3 from amino acid #415 through #421 His-Glu-Leu-Tyr-Val-Ser-Phe,corresponds to the tryptic fragment identified above from which theoligonucleotide probes are designed. When the tryptic sequenceHis-Glu-Leu-Tyr-Val-Ser-Phe-(Ser) described above was identified, it wasnoted to be similar to a sequence found in other BMP proteins forexample the sequence His-Pro-Leu-Tyr-Val-Asp-Phe-Ser found in the bovineand human cartilage/bone protein BMP-2A sequence as described inco-pending U.S. application Ser. No. 179,100, U.S. Pat. No. 5,013,649.BMP-6 therefore represents a new member of the BMP subfamily of TGF-βlike molecules which includes the molecules BMP-2 (also sometimesreferred to as BMP-2A or BMP-2 Class I), 3, 4 (also sometimes referredto as BMP-2B or BMP-2 Class II), 5 and 7 described in co-pendingapplications cited above.

Based on knowledge of other BMP proteins, as well as other proteins inthe TGF-β family, BMP-6 is predicted to be synthesized as a precursormolecule and the precursor polypeptide would be cleaved between aminoacid #381 and amino acid #382 yielding a 132 amino acid maturepolypeptide with a calculated molecular weight of approximately 15 Kd.The mature form of BMP-6 contains three potential N-linked glycosylationsites per polypeptide chain.

It is contemplated that the active BMP-6 protein molecule is a dimer. Itis further contemplated tht the processing of BMP-6 into the mature forminvolves dimerization and removal of the N-terminal region in a manneranalogous to the processing of the related protein TGF-β L. E. Gentry,et al., Molec. & Cell. Biol. 8:4162 (1988); R. Dernyck, et al., Nature316:701 (1985)!.

Comparision of the sequence of murine Vgr-1 Lyons, et al., PNAS 86:4554(1989)! to human BMP-6 reveals a degree of amino acid sequence identitygreater than 92% The murine Vgr-1is likely the murine homologue ofBMP-6. Human BMP-6 shares homology with other BMP molecules as well asother members of the TGF-β superfamily of molecules. The cysteine-richcarboxy-terminal 102 amino acid residues of human BMP-6 shares thefollowing homologies with BMP proteins disclosed in copendingapplications described above: 61% identity with BMP-2; 44% identity withBMP-3, 60% identity with BMP-4; 91% identity with BMP-5; and 87%identity with BMP-7. Human BMP-6 further shares the followinghomologies: 41% identity with TGF-β3; 39% identity with TGF-β2; 37%identity with TGF-β1; 26% identity with Mullerian Inhibiting Substance(MIS), a testicular glycoprotein that causes regression of the Mullerianduct during development of the male embryo; 25% identity with inhibin α;43% identity with inhibin β_(B) ; 49% identity with inhibin β_(A) ; 58%identity with Vg1, a Xenopus factor which may be involved in mesoderminduction in early embryogenesis Weeks and Melton, Cell51:861-867(1987)!; and 59% identity with Dpp the product of theDrosophila decapentaplegic locus which is required for dorsal-ventralspecification in early embryogenesis and is involved in various otherdevelopmental processes at later stages of development Padgett, et al.,Nature 325:81-84 (1987)!.

The procedures described above and additional methods known to thoseskilled in the art may be employed to isolate other related proteins ofinterest by utilizing the bovine or human proteins as a probe source.Such other proteins may find similar utility in, inter alia, fracturerepair, wound healing and tissue repair.

Additional methods known to those skilled in the art may be used toisolate the genetic material encoding human and other species'cartilage/bone proteins of the invention.

EXAMPLE VI Expression of the BMP-6 Proteins

In order to produce bovine, human or other mammalian proteins of theinvention, the DNA encoding it is transferred into an appropriateexpression vector and introduced into mammalian cells or other preferredeukaryotic or prokaryotic hosts by conventional genetic engineeringtechniques. It is contemplated that the preferred expression system forbiologically active recombinant human proteins of the invention will bestably transformed mammalian cells. It is further contemplated that thepreferred mammalian cells will be CHO cells. The transformed host cellis cultured and the BMP-6 proteins expressed thereby are recovered andpurified. The recombinantly expressed BMP-6 proteins are free ofproteinaceous materials with which they ordinarily are associated innature and are purified from other proteinaceous materials with whichthey are co-produced as well as from other contaminants, such asmaterials found in the culture media.

In order to express biologically active human BMP-6 proteins, a selectedhost cell is transformed, using techniques known to those skilled in theart of genetic engineering, with a DNA sequence encoding human BMP-6protein. The DNA encoding BMP-6 comprises nucleotide #1393 to #1698 setforth in FIG. 3 encoding amino acid #412 to #513. The transformed hostcells are cultured and the BMP-6 protein comprising amino acid #412 toamino acid #513 as set forth in FIG. 3 is expressed. The expressedprotein is recovered, isolated and purified form the culture and culturemedium. The purified protein is substantally free from otherproteinaceous materials with which it is co-produced as well as fromother contaminants. In other embodiments, the DNA sequence utilized inexpressing human BMP-6 proteins of the invention comprise the longernucleotide sequence comprising nucleotide #1321 to #1698 encoding theamino acid sequence comprising #388 to #513. In further embodiment theDNA sequence utilized in expression comprises nucleotide #1303 to #1698encoding the amino acid sequence comprising amino acid #382 to #513.

Expression in CHO cells for instance any comprise transformation of thehost cell with a vector containing a DNA sequence comprising nucleotides#160 through #1712 of FIG. 3. The transformed host cell is cultured andthe expressed BMP-6 proteins are recovered and purified. It iscontemplated that the recovered and purified BMP-6 protein compriseswhat is expected to be the mature form comprising amino acid #382-513.However, other forms of BMP-6 may be recovered and purified. These formsinclude proteins comprising amino acid #388-#513 and proteins comprisingamino acid #412 to #513 as set forth in FIG. 3.

One skilled in the art can construct mammalian expression vectors byemploying the DNA sequences of the invention sequences and knownvectors, such as pCD Okayama et al., Mol. Cell Biol., 2:161-170 (1982)!and pJL3, pJL4 Gough et al., EMBO J., 4:645-653 (1985)!. Thetransformation of these vectors into appropriate host cells may resultin expression of the proteins of the invention.

One skilled in the art could manipulate the sequences of the inventionby eliminating or replacing the mammalian regulatory sequences flankingthe coding sequence with bacterial sequences to create bacterial vectorsfor intracellular or extracellular expression by bacterial cells. Thecoding sequences could be further manipulated, for example, ligated toother known linkers or modified by deleting non-coding sequencesthere-from or altering nucleotides therein by other known techniques.The modified coding sequence could then be inserted into a knownbacterial vector using procedures such as described in T. Taniguchi etal., Proc. Natl Acad. Sci. USA, 77:5230-5233 (1980). This exemplarybacterial vector could then be transformed into bacterial host cells anda protein of the invention expressed thereby. For a strategy forproducing extracellular expression of a cartilage and/or bone protein ofthe invention in bacterial cells., see, e.g. European patent applicationEPA 177,343.

Similar manipulations can be performed for the construction of an insectvector See, e.g. procedures described in published European patentapplication 155,476! for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289!.

A method for producing high levels of a protein of the invention frommammalian cells involves the construction of cells containing multiplecopies of the heterologous gene encoding proteins of the invention. Theheterologous gene may be linked to an amplifiable marker, e.g. thedihydrofolate reductase (DHFR) gene for which cells containing increasedgene copies can be selected for propagation in increasing concentrationsof methotrexate (MTX) according to the procedures of Kaufman and Sharp,J. Mol. Biol., 159:601-629 (1982). This approach can be employed with anumber of different cell types.

For example, a plasmid containing a DNA sequence for a protein of theinvention in operative association with other plasmid sequences enablingexpression thereof and the DHFR expression plasmid pAdA26SV(A)3 Kaufmanand Sharp, Mol. Cell. Biol., 2:1304 (1982)! may be co-introduced intoDHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitationand transfection, electroperation or protoplast fusion. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum, and subsequently selected for amplification by growth inincreasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and5 uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).Protein expression should increase with increasing levels of MTXresistance.

Transformants are cloned, and the proteins of the invention arerecovered, isolated, and purified from the culture medium. Biologicallyactive protein expression is monitored by the Rosen-modifiedSampath-Reddi rat bone formation assay described above in Example III.Similar procedures can be followed to produce other related proteins.

EXAMPLE VII Biological Activity of Expressed BMP-6 Proteins

To measure the biological activity of the expressed proteins obtained inExample VI above, the BMP-6 proteins are recovered from the culturemedia and purified. BMP-6 may be partially purified on a HeparinSepharose column. 4 ml of the collected post transfection conditionedmedium supernatant from one 100 mm culture dish is concentratedapproximately 10 fold by ultrafiltration on a YM 10 membrane and thendialyzed against 20 mM Tris, 0.15M NaCl, pH 7.4 (starting buffer). Thismaterial is then applied to a 1.1 ml Heparin Sepharose column instarting buffer. Unbound proteins are removed by an 8 ml wash ofstarting buffer, and bound proteins, including proteins of theinvention, are desorbed by a 3-4 ml wash of 20 mM Tris, 2.0M NaCl, pH7.4.

The proteins bound by the Heparin column are concentrated approximately10-fold on a Centricon 10 and the salt reduced by diafiltration with0.1% trifluoroacetic acid. The appropriate amount of this solution ismixed with 20 mg of rat matrix and then assayed for in vivo bone and/orcartilage formation activity by the Rosen-modified Sampath-Reddi assay.A mock transfection supernatant fractionation is used as a control.

The implants containing rat matrix to which specific amounts of humanBMP-6 proteins of the invention have been added are removed from ratsafter seven days and processed for histological evaluation.Representative sections from each implant are stained for the presenceof new bone mineral with von Kossa and acid fuschin, and for thepresence of cartilage-specific matrix formation using toluidine blue.The types of cells present within the section, as well as the extent towhich these cells display phenotype are evaluated and scored asdescribed in Example III.

Levels of activity may also be tested for host cell extracts. Partialpurification is accomplished in a similar manner as described aboveexcept that 6M urea is included in all the buffers.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

What is claimed is:
 1. A purified human bone morphogenetic protein-6produced by the steps of(a) culturing in a suitable culture medium acell transformed with a DNA sequence comprising the DNA sequence of FIG.3 from nucleotide 1321 to 1698; and (b) recovering from said culturemedium a protein comprising the amino acid sequence from amino acid 388to amino acid 513 as shown in FIG.
 3. 2. A composition comprising apurified BMP-6! bone morphogenetic protein-6 produced by the steps of(a)transforming a host cell with a DNA sequence comprising nucleotides #160to #1712 of FIG. 3; (b) culturing said transformed cell in a suitableculture medium; and (c) isolating and purifying said protein from saidculture medium.
 3. The composition of claim 2 wherein said proteincomprises amino acids #382 to #513 of FIG.
 3. 4. The composition ofclaim 2 wherein said protein is characterized by the ability to inducethe formation of cartilage and/or bone.
 5. A purified bone morphogeneticprotein-6 (BMP-6) comprising an amino acid sequence selected from thegroup consisting of:(a) amino acids 412 through 513 of FIG. 3; (b) aminoacids 388 through 513 of FIG. 3; and (c) amino acids 382 through 513 ofFIG.
 3. 6. The protein of claim 5 wherein said protein is a disulfidelinked dimer wherein at least one of the subunits of said dimercomprises amino acids 382 through 513 of FIG.
 3. 7. The protein of claim5 wherein said protein is a disulfide linked dimer wherein at least oneof the subunits of said dimer comprises amino acids 388 through 513 ofFIG.
 3. 8. The protein of claim 5 wherein said protein is a disulfidelinked dimer wherein at least one of the subunits of said dimercomprises amino acids 412 through 513 of FIG.
 3. 9. A pharnaceuticalcomposition comprising an amount of the BMP-6 protein of claim 5effective to induce the formation of cartilage and/or bone in admixturewith a pharmaceutically acceptable vehicle.
 10. A method for inducingbone and/or cartilage formation in a patient in need of same comprisingadministering to said patient the pharmaceutical composition of claim 9.11. A BMP-6 protein encoded by a DNA sequence comprising a DNA sequenceselected from the group consisting of the DNA molecules of ATCC deposits68245 and
 68021. 12. A BMP-6 protein comprising amino acids 382 through513 of FIG.
 3. 13. A purified BMP-6 polypeptide comprising an amino acidsequence encoded by a DNA sequence comprising a DNA sequence selectedfrom the group consisting of:(a) nucleotides 1393 through 1698 of FIG.3; (b) nucleotides 1321 through 1698 of FIG. 3; (c) nucleotides 1303through 1698 of FIG. 3; and (d) naturally occurring allelic sequencesand degenerative codon sequences of (a) through (c).
 14. A purifiedBMP-6 polypeptide encoded by a DNA sequence which hybridizes understringent wash conditions of 0.1× SSC at 65° C. to a DNA sequenceselected from the group consisting of:(a) nucleotides 1393 through 1698of FIG. 3; (b) nucleotides 1321 through 1698 of FIG. 3; (c) nucleotides1303 through 1698 of FIG. 3; and (d) naturally occurring allelicsequences and degenerative codon sequences of (a) through (c).
 15. Apurified BMP-6 polypeptide comprising an amino acid sequence selectedfrom the group consisting of amino acid sequences encoded by a DNAsequence comprising nucleotides 289 through 666 of FIG. 2.